U.S. patent application number 10/226810 was filed with the patent office on 2003-01-23 for methods of treating conditions associated with corneal injury.
This patent application is currently assigned to XOMA Corporation. Invention is credited to Scannon, Patrick J..
Application Number | 20030017986 10/226810 |
Document ID | / |
Family ID | 24224809 |
Filed Date | 2003-01-23 |
United States Patent
Application |
20030017986 |
Kind Code |
A1 |
Scannon, Patrick J. |
January 23, 2003 |
Methods of treating conditions associated with corneal injury
Abstract
The present invention provides methods of treating a subject
suffering from adverse effects, complications or conditions,
associated with or resulting from a corneal injury including,
corneal infection or ulceration, by topical administration of
suitable ophthalmic preparations of
bactericidal/permeability-increasing (BPI) protein products.
Inventors: |
Scannon, Patrick J.; (San
Francisco, CA) |
Correspondence
Address: |
Janet M. McNicholas, Ph.D
McAndrews, Held & Malloy, Ltd.
34th Floor
500 W. Madison Street
Chicago
IL
60661
US
|
Assignee: |
XOMA Corporation
|
Family ID: |
24224809 |
Appl. No.: |
10/226810 |
Filed: |
August 22, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10226810 |
Aug 22, 2002 |
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09941198 |
Aug 27, 2001 |
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09941198 |
Aug 27, 2001 |
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09677507 |
Oct 2, 2000 |
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09677507 |
Oct 2, 2000 |
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09189040 |
Nov 10, 1998 |
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09189040 |
Nov 10, 1998 |
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08557289 |
Nov 14, 1995 |
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Current U.S.
Class: |
514/2.2 ;
514/12.2; 514/2.4; 514/20.8; 514/3.3; 514/9.4 |
Current CPC
Class: |
A61K 2300/00 20130101;
A61P 27/00 20180101; A61K 38/1751 20130101; A61K 38/1751 20130101;
A61P 27/02 20180101 |
Class at
Publication: |
514/12 |
International
Class: |
A61K 038/17 |
Claims
What is claimed is:
1. A method for treating corneal epithelium injury associated
infection comprising topically administering to the cornea of a
subject having a corneal epithelium injury a
bactericidal/permeability-increasing (BPI) protein product in an
amount effective to reduce hyperemia, chemosis, mucous discharge,
neovascularization or ulcer formation.
2. The method of claim 1 wherein the BPI protein product is an
amino-terminal fragment of BPI protein.
3. The method of claim 1 wherein the BPI protein product is
rBPI.sub.21.
4. The method of claim 1 wherein the BPI protein product is
rBPI.sub.23.
5. The method of claim 1 wherein the BPI protein product is
rBPI.sub.42.
6. The method of claim 1 further comprising administration of a
non-BPI antibiotic or a non-BPI anti-fungal agent.
7. The method of claim 1 further comprising administration of an
anti-inflammatory agent.
Description
BACKGROUND OF THE INVENTION
[0001] The present invention relates generally to methods of
treating a subject suffering from adverse effects, complications or
conditions including infection or ulceration associated with or
resulting from corneal injury from, for example, perforation,
abrasion, chemical burn or trauma injury, by topical administration
of bactericidal/permeability-inc- reasing (BPI) protein
products.
[0002] Corneal infections, microbial keratitis and infectious
corneal ulceration are increasingly prevalent, serious and
sight-threatening ophthalmic diseases. Infectious or microbial
keratitis is an infection of the cornea characterized by an
ulceration of the corneal epithelium associated with an underlying
inflammatory infiltrate of the corneal stroma. Infectious keratitis
is the most serious complication of wearing contact lenses.
Complications of infectious keratitis include sight-threatening
scar formation, scleral involvement, corneal perforation, and even
loss of the eye. Corneal diseases are estimated to involve several
hundred thousand cases of corneal ulcers and about twice that
number of keratitis cases each year in the U.S. alone. Contact lens
wearers, immunocompromised individuals and patients suffering from
dry eye syndrome are among those most at risk to develop such
corneal lesions. In third world countries, this cause of blindness
is second only to cataract formation.
[0003] Microbial keratitis, or infections of the cornea, can be
caused by various bacteria, fungi, viruses, or parasites. Bacteria
are the most common causes, but the frequency of involvement of
different species may vary from one geographic region to another
and may show a shifting pattern over time. Species of bacteria
causing keratitis in the majority of cases are: (1) Micrococcaceae
(Staphylococcus, Micrococcus), (2) Streprococci, (3) Pseudomonas,
and (4) Enterobacteriaceae (Citrobacter, Klebsiella, Enterobacter,
Serratia, Proteus). Historically, the pneumococcus (Streptococcus
pneumoniae) was a major cause, but now other gram-positive
organisms predominate, with Staphylococcus aureus reported to be
the most common cause of microbial keratitis in the northern United
States. Pseudomonas aeruginosa has also become more prevalent as a
cause of keratitis, particularly in association with overnight
contact lens wear. Infections involving the indigenous bacteria of
the conjunctiva and eyelids (Staphylococcus epidermidis,
Corynebacterium and Propionibacterinum species) are reportedly
being seen more frequently, as are other commensal and less
virulent organisms, especially in immunocompromised hosts. The
variety of organisms most commonly seen in bacterial keratitis has
been documented (see, e.g., Liesegang, Bacterial Keratitis, in
Infectious Disease Clinics of North America, Vol. 6, No. 4,
pp.815-829, December, 1992); however, any organism, under
appropriate circumstances, can be a causative agent of corneal
infection and ulceration.
[0004] Corneal infection is usually precipitated by an epithelial
defect resulting from injury (including perforation, abrasion,
chemical burn or trauma injury) to the cornea or from contact lens
wear. Corneal disease patients and patients receiving topical
corticosteroids or with compromised local or systemic defense
mechanisms appear more susceptible to corneal epithelial defects
precipitating infection.
[0005] The cornea is an avascular structure, and has a protective
coating with two layers of mucosubstances, including an adherent
glycocalyx and a mucin layer produced by goblet cells. The intact
corneal epithelium is usually an effective barrier against
infection, although some bacterial organisms, notably Neisseria
gonorrhoeae and Corynebacterium diphtheriae, can penetrate the
intact epithelium.
[0006] The lids and eyelashes normally harbor microorganisms and
shed them onto the cornea, but the eyelids provide a defensive
system for the cornea, primarily through the lacrimal secretions
and the ocular blink reflex.
[0007] The tear film provides-lubrication to flush away any
organisms or debris. The tear film also contains several
antimicrobial substances, including lysozyme, lactoferrin,
beta-lysins, and complement components, as well as immunoglobulins
(especially secretory IgA) and lymphocytes, which provide a local
defense mechanism. Lactoferrin can enhance the effect of surface
antibodies or inhibit bacterial growth or invasiveness by chelating
iron. Tear lysozyme can directly lyse bacterial cell walls, and
beta-lysins can lyse bacterial membranes. Secretory IgA blocks the
adhesion of bacteria to membranes. Malposition of the lids and
lashes, however, or difficulty in lid closure interferes with these
protective functions and predisposes to corneal infection.
[0008] Predisposing factors to corneal infection therefore include:
(1) trauma or injury (e.g., foreign body, contact lens wear); (2)
abnormal tear function (e.g., dry eye, lacrimal obstruction) and
abnormal lid structure and function (e.g., blepharitis, laopthalmus
entropion, ectropion, trichiasis); (3) corneal diseases (e.g.,
corneal edema); and (4) systemic conditions (e.g., Sjogren's
syndrome, alcoholism, diabetes, rheumatoid arthritis, debilitating
disease, tracheal intubation, central nervous system disease and
psychiatric disturbances, extensive bums, acquired immunodeficiency
syndrome (AIDS), and corticosteroid and immunosuppressive
therapy).
[0009] Contact lens wear is a significant risk factor compromising
the structural integrity of the corneal epithelium and predisposing
toward corneal infection. Contact lens wear give rise to corneal
hypoxia, increased corneal temperature, decreased tear flow to the
cornea, and also provides a constant source of microtrauma to the
corneal epithelium. Soft contact lenses become coated with mucus
and protein after only a few hours of wear, and this may further
enhance the adherence of bacteria. Hard gas-permeable lenses, daily
wear soft contact lenses, extended wear soft contact lenses,
therapeutic soft contact lenses, and disposable contact lenses all
increase the risk of microbial keratitis. Overnight wear,
especially after cataract surgery, is associated with the highest
risk. Other factors contributing to contact lens-associated
microbial keratitis include the failure to follow proper contact
lens wear instructions, poor contact lens hygiene, use of
contaminated lens solutions, and microtrauma at the time of the
insertion and removal. Pseudomonas aeruginosa and Staphylococcus
are the most common organisms isolated in contact lens-associated
keratitis.
[0010] Acanthamoeba keratitis, a parasitic infection, has been
linked to prolonged exposure to contaminated water, especially in
contact lens wearers and in individuals who use hot tubs or
swimming pools. Fungal keratitis is seen in different clinical
situations. Filamentary fungal keratitis is seen after injury to
the cornea in agricultural settings, whereas yeast keratitis is
seen in any environment in patients who are immunocompromised or
have a severely damaged cornea.
[0011] The severity of the bacterial keratitis depends, for the
most part, on the virulence of the invading bacteria but also is
correlated to the previous health of the cornea and the host
response. The pathogenicity of particular organisms is correlated
with the ability to adhere to the edge or base of an epithelial
defect and to invade the corneal stroma. Pseudomonas aeruginosa,
Staphylococcus aureus, and Streptococcus pneumoniae adhere tightly
to the edge of an epithelial defects, probably because of membrane
appendages called fibrillae (in gram-positive organisms) or
fimbriae (in gram-negative organisms). Specific adhesions on the
surface of these appendages may interact with specific receptors on
the corneal epithelium. Some species, notably Pseudomonas and
Staphylococcus, produce an extracellular polysaccharide slime layer
which may have a role in adherence to a variety of surfaces,
especially soft contact lenses. The mechanisms of penetration of
bacteria into the corneal stroma following entry through an
epithelial injury are poorly understood but are probably correlated
with the production of toxins and enzymes. Pseudomonas and Serratia
species have proteoglycanase (e.g., collagenase) activity that can
liquify the stroma. Other organisms have other properties that
permit adherence and corneal destruction. The host's
polymorphonuclear response to the infection contributes to the
tissue destruction and collagen breakdown as a result of lysozymal
enzymes and other proteases.
[0012] In a previously healthy cornea, the presence of a corneal
epithelial ulceration with adherent mucopurulent exudate and
inflammatory cells in the adjacent corneal stroma and the anterior
chamber should lead to a presumptive diagnosis of bacterial
keratitis. The eyelids may be stuck together and the tear film
filled with inflammatory cells. Nonspecific symptoms include
decreased vision, redness, pain, conjunctival and lid swelling and
a discharge. Clinical signs may include increasing stromal edema,
hypopyon, iris miosis, and synechiae.
[0013] In a patient with a cornea previously damaged by herpes
simplex virus infection, corneal edema, or trauma, it may be
difficult to distinguish the clinical signs of infection from the
residua of the underlying structural abnormalities. A bacterial
infection should be suspected when there is an increase in the
extent of epithelial or stromal ulceration or anterior chamber
inflammation. Antecedent therapy with systemic or local ocular
immunosuppressive agents, especially corticosteroids, not only
increases the risk of ocular infection but may alter the clinical
response in such a way as to mask or alter some of the typical
features of infection.
[0014] There are difficulties in distinguishing bacterial keratitis
from other forms of microbial keratitis or from the multiple
noninfectious causes of corneal ulceration. The differential
diagnosis includes fungal, viral, and parasitic keratitis as well
as toxic or chemical keratopathy, indolent or neurotrophic
ulceration, severe dry eyes, and various other insults to the
cornea. The history, physical examination, and evidence of the
onset of the new disease process may permit a presumptive
diagnosis. When corneal infection is suspected, the culture
strategy may include screening for the most likely agents: aerobic
bacteria, anaerobic bacteria, filamentous fungi, and yeasts. A
corneal sample may be obtained by scraping, using the magnification
of the slit lamp biomicroscope, and topical anesthesia. With deep
keratitis, fragments of the cornea may be excised with a
microsurgical scissor or trephine. More than one species of microbe
may be present in a corneal infection. Negative cultures are not
uncommon in cases of suspected infectious corneal ulcers, and may
be due to inadequate sampling methods, the improper selection of
media, prior antibiotic treatment, or improper interpretation of
data.
[0015] Currently, the initial therapy for suspected microbial
keratitis is based on the severity of the keratitis and a
familiarity with the most likely causative organisms. Suspected
microbial keratitis is typically treated as a bacterial ulcer until
a more definitive laboratory diagnosis is made. Initial antibiotic
therapy may be based on the results of the Gram stain or Giemsa
stain, or a broad spectrum antibiotic may be administered as the
initial treatment, especially in cases of serious suspected
microbial keratitis. Most U.S. practitioners are not willing to
leave the lesion untreated while waiting for culture results.
Generally, a broad spectrum antibiotic is prescribed following
examination. Such initial antibiotic therapy may be modified after
the causative organism is identified from correlation of the Gram
stain, culture results, and the clinical response. There are a
relatively small number of antibiotics available commercially as
topical ophthalmic preparations. Many other antibiotics can be
prepared for topical ophthalmic use in treating serious corneal
infections, however, their use is expensive and inconvenient, and
many are not well tolerated or have limited antibacterial spectra.
Pseudomonas species account for many serious, and rapidly
destructive, corneal infections. In fact, ocular disease produced
by the opportunistic bacterial pathogen P. aeruginosa often leads
to a fulminating and highly destructive infection resulting in
rapid liquefaction of the cornea and blindness. Antibiotic
treatment is not always successful due to the resistance of many
clinical strains. The patient is vulnerable during the ulcerative
period to sequelae that are sight threatening and even could create
a situation where the eye had to be enucleated. Any agent that
could accelerate the healing time, for example, would be highly
desired by medical practitioners. Thus, there is an unmet need to
develop agents with therapeutic efficacy, either alone or in
conjunction with existing agents, against these organisms.
[0016] In cases where there is the need for frequent administration
of antimicrobial drops and the need to examine the patient daily,
patients may be hospitalized. Patient isolation is not usually
necessary, although contact with preoperative patients should be
avoided. Outpatient therapy may be preferred for compliant patients
or those with milder disease.
[0017] The ideal topical antibiotic agent should be bactericidal at
reasonable concentrations against the corneal pathogens, should be
able to penetrate the cornea, and should be free of significant
adverse affects. Factors considered in the use of systemic
antibiotics (i.e., achievable serum levels, distribution space, and
absorption and excretion characteristics) are not applicable. Some
patients may respond to commercial-strength topical antibiotic
agents given at frequent intervals, but fortified topical
antibiotic agents are usually more effective. For example, recent
fluoroquinolone antibiotics, norfloxacin and ciprofloxacin, may be
effective at commercial strength for infections by susceptible
bacteria. Drug penetration into the cornea may be increased with
higher concentration of the drug, more frequent application, longer
contact time with the use of some vehicles, with more lipophilic
antibiotic agents, and with the absence of the epithelium.
Solutions may be preferred to ointments because of the flexibility
in varying the concentration and the ease of administration. A
fortified topical antibiotic agent may be prepared by adding the
desired amount of the parenteral antibiotic to an artificial tear
solution.
[0018] The primary goal of current therapy is to administer an
antibiotic which will be effective quickly without causing
significant ocular and systemic toxicity. Other considerations or
goals are to reduce the corneal inflammatory response, to limit
structural corneal damage, and to promote corneal
reepithelialization. As is the case in other organ systems, healing
of a corneal ulcer is often accompanied by neovascularization. In
the eye, neovascularization and scarring are particularly
deleterious as vision is dependent upon a clear cornea which
requires the maintenance of the highly organized fibrin structure.
Immunosuppressant corticosteroids can be used to inhibit the vessel
formation but many ophthalmologists would rather not risk this
indiscriminate type of immune suppression while the cornea is
vulnerable due to ulceration. Thus, there exists a need in the art
for agents with therapeutic efficacy in reduction of
neovascularization and scanning but without the generalized immune
suppressing effects of steroids.
[0019] Even with current antibiotic and steroid therapies, major
concerns regarding the treatment of infectious corneal ulcers
remain, including: broad spectrum application; fear of antibiotic
resistant strains of microbes; controversy regarding prophylactic
versus therapeutic treatment of suspected infectious ulcers;
non-compliant patients; control of neovascularization and scar
formation. There exists a need for new therapeutic agents that
would better address these issues.
[0020] BPI is a protein isolated from the granules of mammalian
polymorphonuclear leukocytes (PMNs or neutrophils), which are blood
cells essential in the defense against invading microorganisms.
Human BPI protein has been isolated from PMNs by acid extraction
combined with either ion exchange chromatography [Elsbach, J. Biol.
Chem., 254:11000 (1979)] or E. coli affinity chromatography [Weiss,
et al., Blood, 69:652 (1987)]. BPI obtained in such a manner is
referred to herein as natural BPI and has been shown to have potent
bactericidal activity against a broad spectrum of gram-negative
bacteria. The molecular weight of human BPI is approximately 55,000
daltons (55 kD). The amino acid sequence of the entire human BPI
protein and the nucleic acid sequence of DNA encoding the protein
have been reported in FIG. 1 of Gray et al., J. Biol. Chem.,
264:9505 (1989), incorporated herein by reference. The Gray et al.
amino acid sequence is set out in SEQ ID NO: 1 hereto.
[0021] BPI is a strongly cationic protein. The N-terminal half of
BPI accounts for the high net positive charge; the C-terminal half
of the molecule has a net charge of -3. [Esbach and Weiss (1981),
supra.] A proteolytic N-terminal fragment of BPI having a molecular
weight of about 25 kD has an amphipathic character, containing
alternating hydrophobic and hydrophilic regions. This N-terminal
fragment of human BPI possesses the anti-bacterial efficacy of the
naturally-derived 55 kD human BPI holoprotein. [Ooi et al., J. Bio.
Chem., 262: 14891-14894 (1987)]. In contrast to the N-terminal
portion, the C-terminal region of the isolated human BPI protein
displays only slightly detectable anti-bacterial activity against
gram-negative organisms. [Ooi et al., J. Exp. Med., 174:649
(1991).] An N-terminal BPI fragment of approximately 23 kD,
referred to as "rBPI.sub.23," has been produced by recombinant
means and also retains anti-bacterial activity against
gram-negative organisms. Gazzano-Santoro et al., Infect. Immun.
60.4754-4761 (1992).
[0022] There continues to exist a need in the art for new methods
and materials for treatment of corneal injury, including infection
or ulceration. Products and methods responsive to this need would
ideally involve substantially non-toxic, non-irritating ophthalmic
preparations available in suitable amounts by means of synthetic or
recombinant methods. Ideal compounds would be capable of
penetrating corneal tissue and would prevent or reduce the number
and severity of adverse effects, complications or conditions
associated with or resulting from cornea injury. Alternatively, or
in addition, such ideal compounds would enhance the effect of, or
reduce the need for, other concurrently administered
anti-inflammatory and/or antimicrobial therapeutic agents.
SUMMARY OF THE INVENTION
[0023] The present invention provides novel methods of treating
corneal epithelial injury associated infection comprising topical
application to the cornea of a subject having a corneal epithelial
injury a bactericidal/permeability-increasing (BPI) protein product
in an amount effective to reduce hyperemia, chemosis,
neovascularization, mucous discharge or ulcer formation. Methods
according to the invention are thus useful for reducing the adverse
effects, complications or conditions associated with or resulting
from a corneal injury including, corneal infection or ulceration,
by topically administering a therapeutically effective amount of an
ophthalmic preparation of a BPI protein product to a subject
suffering from the effects of such corneal infection, ulceration or
injury. The invention derives in part from the surprising discovery
that topically administered BPI protein products penetrate the
cornea and prevent or reduce adverse effects associated with
corneal infections and ulcerations. These adverse effects include
hyperemia, chemosis, mucous discharge, tearing, photophobia,
keratitis, neovascularization, ulcer formation, opacification
(clouding), contrast sensitivity, scarring, pain or loss of visual
acuity. Confirmation of beneficial effects of practice of the
invention is provided by standard ophthalmological examination
including, for example, slit lamp biomicroscopy.
[0024] Methods of the present invention contemplate administration
of a BPI protein product in ophthalmologically acceptable
preparations which may include, or be concurrently administered
with, anti-inflammatory agents such as corticosteroids and/or
antimicrobial agents such as ciprofloxacin gentamicin, ofloxacin
and anti-fungal agents. Presently preferred BPI protein products of
the invention include biologically active amino terminal fragments
of the BPI holoprotein, recombinant products such as rBPI.sub.21
and rBPI.sub.42 and recombinant or chemically synthesized
BPI-derived peptides as described in detail below.
[0025] The invention further provides for the use of a BPI protein
products for manufacture of a topical medicament for reducing the
above-noted adverse effects, complications or conditions,
associated with or resulting from corneal infection and
ulceration.
[0026] Numerous additional aspects and advantages of the invention
will become apparent to those skilled in the art upon considering
the following detailed description of the invention, which
describes the presently preferred embodiments thereof, reference
being made to the drawing wherein:
[0027] FIG. 1 is a photograph of a "control" rabbit eye 72 hours
after corneal epithelium puncture and injection with Pseudomonas
aeruginosa wherein post-injection treatments included an ophthalmic
product vehicle solution only; and
[0028] FIG. 2 is a photograph of a rabbit eye 72 hours after
corneal epithelium puncture and injection with Pseudomonas
aeruginosa wherein the cornea was treated according to the present
invention.
DETAILED DESCRIPTION OF THE INVENTION
[0029] Incorporated by reference herein are the disclosures of the
applicant's co-owned, co-pending, concurrently-filed U.S. patent
application Ser. No. ______ (Attorney Docket No. 27129/33007)
entitled "Methods of Treating Conditions Associated With Corneal
Transplantation."
[0030] The present invention relates to the surprising discovery
that a bactericidal/permeability-increasing (BPI) protein product
can be topically administered to the cornea, in an amount effective
to reduce hyperemia, chemosis, neovascularization, mucous discharge
or ulcer formation associated with or resulting from corneal
epithelial injury associated infection. Methods according to the
invention are useful for treating subjects suffering from corneal
infection, ulceration, or injury, and conditions associated
therewith or resulting therefrom. Particularly valuable is the lack
of corneal tissue toxicity and the effectiveness of such topically
administered BPI protein products, given that penetration of
corneal tissue is a necessary but not sufficient step for
therapeutic efficacy. BPI protein products are shown herein to
prevent or reduce adverse effects of corneal injury associated
infection and ulceration including, for example, preventing or
reducing hyperemia, chemosis, mucous discharge, tearing,
photophobia, keratitis, neovascularization, ulcer formation (i.e.,
prevent ulcer development or reduce ulcer size) opacification
(clouding), contrast sensitivity, scanning, pain and loss of visual
acuity as measured by standard ophthalmological examination, using,
slit lamp biomicroscopy to note clinical manifestations.
[0031] According to one aspect of the invention, suitable
ophthalmic preparations of BPI protein product alone, in an amount
sufficient for monotherapeutic effectiveness, may be administered
to a subject suffering from corneal infection, ulceration, or
injury, and conditions associated therewith or resulting therefrom.
When used to describe administration of BPI protein product alone,
the term "amount sufficient for monotherapeutic effectiveness"
means a suitable ophthalmic preparation having an amount of BPI
protein product that provides beneficial effects, including
anti-microbial and/or anti-angiogenic effects, when administered as
a monotherapy. The invention utilizes any of the large variety of
BPI protein products known to the art including natural BPI protein
isolates, recombinant BPI protein, BPI fragments, BPI analogs, BPI
variants, and BPI-derived peptides.
[0032] According to another aspect of the invention, a patient may
be treated by concurrent administration of suitable ophthalmic
preparations of a BPI protein product in an amount sufficient for
combinative therapeutic effectiveness and one or more
immunosuppressant corticosteroids in amounts sufficient for
combinative therapeutic effectiveness. This aspect of the invention
contemplates concurrent administration of BPI protein product with
any corticosteroid or combinations of corticosteroids, including
prednisolone and dexamethasone and contemplates that, where
corticosteroid therapy is required, lesser amounts will be needed
and/or that there will be a reduction in the duration of
treatment.
[0033] According to another aspect of the invention, a subject
suffering from corneal epithelial injury associated infection or
ulceration, and conditions associated therewith or resulting
therefrom, may be treated by concurrent administration of suitable
ophthalmic preparations of a BPI protein product in an amount
sufficient for combinative therapeutic effectiveness and one or
more antibiotics in amounts sufficient for combinative therapeutic
effectiveness. This aspect of the invention contemplates concurrent
administration of BPI protein product with any antimicrobial agent
or combinations thereof for topical use in the eye including:
antibacterial agents such as gentamicin, tobramycin, bacitracin,
chloramphenicol, ciprofloxacin, ofloxacin, norfloxacin,
erythromycin, bacitracin/neomycin/polymyxin B, sulfisoxazole,
sulfacetamide, tetracycline, polymyxin/bacitracin,
trimethroprim/polymyxin B, vancomycin, clindamycin, ticarcillin,
penicillin, oxacillin or cefazolin; antifungal agents such as
amphotericin B, nystatin, natamycin (pimaricin), miconazole,
ketocanozole or fluconazole; antiviral agents such as idoxuridine,
vidarabine or trifluridine; and antiprotozoal agents such as
propamidine, neomycin, clotrimazol, miconazole, itraconazole or
polyhexamethylene biguanide.
[0034] This aspect of the invention is based on the improved
therapeutic effectiveness of suitable ophthalmic preparations of
BPI protein products with antibiotics, e.g., by increasing the
antibiotic susceptibility of infecting organisms to a reduced
dosage of antibiotics providing benefits in reduction of cost of
antibiotic therapy and/or reduction of risk of toxic responses to
antibiotics. BPI protein products may lower the minimum
concentration of antibiotics needed to inhibit in vitro growth of
organisms at 24 hours. In cases where BPI protein product does not
affect growth at 24 hours, BPI protein product may potentiate the
early bactericidal effect of antibiotics in vitro at 0-7 hours. The
BPI protein products may exert these effects even on organisms that
are not susceptible to the direct bactericidal or growth inhibitory
effects of BPI protein product alone.
[0035] This aspect of the invention is correlated to effective
reversal of the antibiotic resistance of an organism by
administration of a BPI protein product and antibiotic. BPI protein
products may reduce the minimum inhibitory concentration of
antibiotics from a level within the clinically resistant range to a
level within the clinically susceptible range. BPI protein products
thus may convert normally antibiotic-resistant organisms into
antibiotic-susceptible organisms.
[0036] According to these aspects of the invention, suitable
ophthalmic preparations of the BPI protein product along with
corticosteroids and/or antibiotics are concurrently administered in
amounts sufficient for combinative therapeutic effectiveness. When
used to describe administration of a suitable ophthalmic
preparation of BPI protein product in conjunction with a
corticosteroid, the term "amount sufficient for combinative
therapeutic effectiveness" with respect to the BPI protein product
means at least an amount effective to reduce or minimize
neovascularization and the term "amount sufficient for combinative
therapeutic effectiveness" with respect to a corticosteroid means
at least an amount of the corticosteroid that reduces or minimizes
inflammation when administered in conjunction with that amount of
BPI protein product. Either the BPI protein product or the
corticosteroid, or both, may be administered in an amount below the
level required for monotherapeutic effectiveness against adverse
effects associated with or resulting from corneal injury associated
infection/ulceration. When used to describe administration of a
suitable ophthalmic preparation of BPI protein product in
conjunction with an antimicrobial, the term "amount sufficient for
combinative therapeutic effectiveness" with respect to the BPI
protein product means at least an amount effective to reduce
neovascularization and/or increase the susceptibility of the
organism to the antimicrobial, and the term "amount sufficient for
combinative therapeutic effectiveness" with respect to an
antimicrobial means at least an amount of the antimicrobial that
produces bactericidal or growth inhibitory effects when
administered in conjunction with that amount of BPI protein
product. Either the BPI protein product or the antimicrobial, or
both, may be administered in an amount below the level required for
monotherapeutic effectiveness.
[0037] BPI protein product may be administered in addition to
standard therapy and is preferably incorporated into the care given
the patient exposed to risk of corneal epithelium injury or
actually suffering such injury. Treatment with BPI protein product
is preferably continued for at least 1 to 30 days, and potentially
longer if necessary, in dosage amounts (e.g., dropwise
administration of about 10 to about 200 .mu.L solution of a BPI
protein product at about 1 to 2 mg/mL) determined by good medical
practice based on the clinical condition of the individual
patient.
[0038] Suitable ophthalmic preparations of BPI protein products may
provide benefits as a result of their ability to neutralize heparin
and their ability to inhibit heparin-dependent angiogenesis. The
anti-angiogenic properties of BPI have been described in Little et
al., co-owned, co-pending U.S. application Ser. No. 08/435,855 and
co-owned U.S. Pat. No. 5,348,942, both incorporated by reference
herein.
[0039] Suitable ophthalmic preparations of BPI protein products may
provide additional benefits as a result of their ability to
neutralize endotoxin associated with gram-negative bacteria and/or
endotoxin released by antibiotic treatment of patients with corneal
infection/ulceration. Suitable ophthalmic preparations of BPI
protein products could provide further benefits due to their
anti-bacterial activity against susceptible bacteria and fungi, and
their ability to enhance the therapeutic effectiveness of
antibiotics and anti-fungal agents. See, e.g., Horwitz et al.,
co-owned, co-pending U.S. application Ser. No. 08/372,783, filed
Jan. 13, 1995 as a continuation-in-part of U.S. application Ser.
No. 08/274,299, filed Jul. 11, 1994, which are all incorporated
herein by reference and which describe BPI protein product activity
in relation to gram-positive bacteria; and Little et al., co-owned,
co-pending U.S. application Ser. No. 08/372,105, filed Jan. 13,
1995 as a continuation-in-part of U.S. application Ser. No.
08/273,540, filed Jul. 11, 1994, which are all incorporated herein
by reference and which describe BPI protein product activity in
relation to fungi.
[0040] For ophthalmic uses as described herein, the BPI protein
product is preferably administered topically, to the corneal wound
or injury. Topical routes include administration preferably in the
form of ophthalmic drops, ointments, gels or salves. Other topical
routes include irrigation fluids (for, e.g., irrigation of wounds).
Those skilled in the art can readily optimize effective ophthalmic
dosages and administration regimens for the BPI protein
products.
[0041] As used herein, "BPI protein product" includes naturally and
recombinantly produced BPI protein; natural, synthetic, and
recombinant biologically active polypeptide fragments of BPI
protein; biologically active polypeptide variants of BPI protein or
fragments thereof, including hybrid fusion proteins and dimers;
biologically active polypeptide analogs of BPI protein or fragments
or variants thereof, including cysteine-substituted analogs; and
BPI-derived peptides. The BPI protein products administered
according to this invention may be generated and/or isolated by any
means known in the art. U.S. Pat. No. 5,198,541, the disclosure of
which is incorporated herein by reference, discloses recombinant
genes encoding and methods for expression of BPI proteins including
recombinant BPI holoprotein, referred to as rBPI.sub.50 or
rBPI.sub.55 and recombinant fragments of BPI. Co-owned, copending
U.S. patent application Ser. No. 07/885,501 and a
continuation-in-part thereof, U.S. patent application Ser. No.
08/072,063 filed May 19, 1993 and corresponding PCT Application No.
93/04752 filed May 19, 1993, which are all incorporated herein by
reference, disclose novel methods for the purification of
recombinant BPI protein products expressed in and secreted from
genetically transformed mammalian host cells in culture and
discloses how one may produce large quantities of recombinant BPI
products suitable for incorporation into stable, homogeneous
pharmaceutical preparations.
[0042] Biologically active fragments of BPI (BPI fragments) include
biologically active molecules that have the same or similar amino
acid sequence as a natural human BPI holoprotein, except that the
fragment molecule lacks amino-terminal amino acids, internal amino
acids, and/or carboxy-terminal amino acids of the holoprotein.
Nonlimiting examples of such fragments include a N-terminal
fragment of natural human BPI of approximately 25 kD, described in
Ooi et al., J. Exp. Med., 174:649 (1991), and the recombinant
expression product of DNA encoding N-terminal amino acids from 1 to
about 193 or 199 of natural human BPI, described in Gazzano-Santoro
et al., Infect. Immun. 60:4754-4761 (1992), and referred to as
rBPI.sub.23. In that publication, an expression vector was used as
a source of DNA encoding a recombinant expression product
(rBPI.sub.23) having the 31-residue signal sequence and the first
199 amino acids of the N-terminus of the mature human BPI, as set
out in FIG. 1 of Gray et al., supra, except that valine at position
151 is specified by GTG rather than GTC and residue 185 is glutamic
acid (specified by GAG) rather than lysine (specified by AAG).
Recombinant holoprotein (rBPI) has also been produced having the
sequence (SEQ ID NOS: 1 and 2) set out in FIG. 1 of Gray et al.,
supra, with the exceptions noted for rBPI.sub.23, and with the
exception that residue 417 is alanine (specified by GCT) rather
than valine (specified by GTT). Other examples include dimeric
forms of BPI fragments, as described in co-owned and co-pending
U.S. Pat. No. 5,447,913 the disclosures of which are incorporated
herein by reference. Preferred dimeric products include dimeric BPI
protein products wherein the monomers are amino-terminal BPI
fragments having the N-terminal residues from about 1 to 175 to
about 1 to 199 of BPI holoprotein. A particularly preferred dimeric
product is the dimeric form of the BPI fragment having N-terminal
residues 1 through 193, designated rBPI.sub.42 dimer.
[0043] Biologically active variants of BPI (BPI variants) include
but are not limited to recombinant hybrid fusion proteins,
comprising BPI holoprotein or biologically active fragment thereof
and at least a portion of at least one other polypeptide, and
dimeric forms of BPI variants. Examples of such hybrid fusion
proteins and dimeric forms are described by Theofan et al. in
co-owned, copending U.S. patent application Ser. No. 07/885,911,
and a continuation-in-part application thereof, U.S. patent
application Ser. No. 08/064,693 filed May 19, 1993 and
corresponding PCT Application No. US93/04754 filed May 19, 1993,
which are all incorporated herein by reference and include hybrid
fusion proteins comprising, at the amino-terminal end, a BPI
protein or a biologically active fragment thereof and, at the
carboxy-terminal end, at least one constant domain of an
immunoglobulin heavy chain or allelic variant thereof.
[0044] Biologically active analogs of BPI (BPI analogs) include but
are not limited to BPI protein products wherein one or more amino
acid residues have been replaced by a different amino acid. For
example, co-owned, U.S. Pat. No. 5,420,019 and corresponding PCT
Application No. US94/01235 filed Feb. 2, 1994, the disclosures of
which are incorporated herein by reference, discloses polypeptide
analogs of BPI and BPI fragments wherein a cysteine residue is
replaced by a different amino acid. A preferred BPI protein product
described by this application is the expression product of DNA
encoding from amino acid 1 to approximately 193 (particularly
preferred) or 199 of the N-terminal amino acids of BPI holoprotein,
but wherein the cysteine at residue number 132 is substituted with
alanine and is designated rBPI.sub.21.DELTA.cys or rBPI.sub.21.
Other examples include dimeric forms of BPI analogs; e.g. co-owned
and co-pending U.S. patent application Ser. No. 08/212,132 filed
Mar. 11, 1994, the disclosures of which are incorporated herein by
reference.
[0045] Other BPI protein products useful according to the methods
of the invention are peptides derived from or based on BPI produced
by synthetic or recombinant means (BPI-derived peptides), such as
those described in PCT Application No. US95/09262 filed Jul. 20,
1995 corresponding to co-owned and copending U.S. application Ser.
No. 08/504,841 filed Jul. 20, 1995, PCT Application No. US94/10427
filed Sep. 15, 1994, which corresponds to U.S. patent application
Ser. No. 08/306,473 filed Sep. 15, 1994, and PCT Application No.
US94/02465 filed Mar. 11, 1994, which corresponds to U.S. patent
application Ser. No. 08/209,762, filed Mar. 11, 1994, which is a
continuation-in-part of U.S. patent application Ser. No.
08/183,222, filed Jan. 14, 1994, which is a continuation-in-part of
U.S. patent application Ser. No. 08/093,202 filed Jul. 15, 1993
(for which the corresponding international application is PCT
Application No. US94/02401 filed Mar. 11, 1994), which is a
continuation-in-part of U.S. patent application Ser. No. 08/030,644
filed Mar. 12, 1993, the disclosures of all of which are
incorporated herein by reference.
[0046] The safety of BPI protein products for systemic
administration to humans has been established healthy volunteers
and in human experimental endotoxemia studies published in von der
Mohlen et al., Blood, 85(12):3437-3343 (1995) and von der Mohlen et
al., J. Infect. Dis., 172:144-151 (1995).
[0047] Presently preferred BPI protein products include
recombinantly-produced N-terminal fragments of BPI, especially
those having a molecular weight of approximately between 21 to 25
kD such as rBPI.sub.21 or rBPI.sub.23; or dimeric forms of these
N-terminal fragments (e.g., rBPI.sub.42 dimer). Additionally,
preferred BPI protein products include rBPI.sub.55 and BPI-derived
peptides. Presently most preferred is the rBPI.sub.21 protein
product.
[0048] The administration of BPI protein products is preferably
accomplished with a pharmaceutical composition comprising a BPI
protein product and a pharmaceutically acceptable diluent,
adjuvant, or carrier. The BPI protein product may be administered
without or in conjunction with known surfactants, other
chemotherapeutic agents or additional known antimicrobial agents.
Presently preferred pharmaceutical compositions containing BPI
protein products (i.e., rBPI.sub.21,) comprise the BPI protein
product at a concentration of 2 mg/ml in 5 mM citrate, 150 mM NaCl,
0.2% poloxamer 403 (Pluronic P123, BASF Wyandotte, Parsippany,
N.J.) (most preferred) or 0.2% poloxamer 333 (Pluronic P103 BASF
Wyandotte, Parsippany, N.J.) and 0.002% polysorbate 80 (Tween 80,
ICI Americas Inc., Wilmington, Del.). Compositions of BPI protein
product and anti-bacterial activity-enhancing poloxamer surfactants
are described in co-owned, co-pending U.S. patent application Ser.
No. 08/372,104 filed Jan. 13, 1995 and 08/530,599 filed Sep. 19,
1995 the disclosures of which are incorporated herein by reference.
Another pharmaceutical composition containing BPI protein products
(i.e., rBPI.sub.21) comprises the BPI protein product at a
concentration of 2 mg/ml in 5 mM citrate, 150 mM NaCl, 0.2%
poloxamer 188 (Pluronic F 68, BASF Wyandotte, Parsippany, N.J.) and
0.002% polysorbate 80. Yet another pharmaceutical composition
containing BPI protein products (e.g., rBPI.sub.55, rBPI.sub.42,
rBPI.sub.23) comprises the BPI protein product at a concentration
of 1 mg/ml in citrate buffered saline (5 or 20 mM citrate, 150 mM
NaCl, pH 5.0) comprising 0.1% by weight of poloxamer 188 (Pluronic
F-68, BASF Wyandotte, Parsippany, N.J.) and 0.002% by weight of
polysorbate 80 (Tween 80, ICI Americas Inc., Wilmington, Del.).
Such combinations are described in co-owned, co-pending PCT
Application No. US94/01239 filed Feb. 2, 1994, which corresponds to
U.S. patent application Ser. No. 08/190,869 filed Feb. 2, 1994 and
U.S. patent application Ser. No. 08/012,360 filed Feb. 2, 1993, the
disclosures of all of which are incorporated herein by
reference.
[0049] Other aspects and advantages of the present invention will
be apparent upon consideration of the following illustrative
examples wherein: Example 1 addresses the effects of various BPI
protein products with respect to Pseudomonas infection in a corneal
infection/ulceration rabbit model; Example 2 addresses the effects
of varying formulations of a single BPI protein product with
respect to Pseudomonas infection in a corneal infection/ulceration
rabbit model; Example 3 addresses the effects of BPI protein
product administration on Pseudomonas infection in a corneal
infection/ulceration rabbit model either alone and in
co-administration with various antibiotics.
EXAMPLE 1
Effect of BPI Protein Products on Pseudomonas Infection in a
Corneal Ulceration Rabbit Model
[0050] The effects of various BPI protein products were first
evaluated in the context of administration both prior to and after
Pseudomonas infection in a corneal infection/ulceration rabbit
model. BPI protein products tested included: rBPI.sub.42 (Expt. 1),
rBPI.sub.21, in a formulation with poloxamer 188 (Expt. 2), an
anti-angiogenic BPI-derived peptide designated XMP.112 (Expt. 3),
an anti-bacterial BPI-derived peptide designated XMP.105 (Expt. 4)
and rBPI.sub.21 in a formulation with poloxamer 403 (Expt. 5). The
structure of XMP.112 and XMP.105 are set out in previously-noted
PCT Application No. 94/02465.
[0051] For these experiments, the infectious organism was a strain
of Pseudomonas aeruginosa 19660 obtained from the American Type
Culture Collection (ATCC, Rockville, Md.). The freeze dried
organism was resuspended in nutrient broth (Difco, Detroit, Mich.)
and grown at 37.degree. C with shaking for 18 hours. The culture
was centrifuged following the incubation in order to harvest and
wash the pellet. The washed organism was Gram stained in order to
confirm purity of the culture. A second generation was cultured
using the same techniques as described above. Second generation
cell suspensions were diluted in nutrient broth and adjusted to an
absorbance of 1.524 at 600 nm, a concentration of approximately
6.55.times.10.sup.9 CFU/ml. A final 1.3.times.10.sup.6 fold
dilution in nutrient broth yielded 5000 CFU/mL or
1.0.times.10.sup.2 CFU/0.02 mL. Plate counts for CFU determinations
were made by applying 100 .mu.L of the diluted cell suspension to
nutrient agar plates and incubating them for 24-48 hours at
37.degree. C.
[0052] For these experiments, the animals used were New Zealand
White rabbits, maintained in rigid accordance to both SERI
guidelines and the ARVO Resolution on the Use of Animals in
Research. A baseline examination of all eyes was conducted prior to
injection in order to determine ocular health. All eyes presented
with mild diffuse fluorescein staining, characteristically seen in
the normal rabbit eye. The health of all eyes fell within normal
limits. Rabbits weighing between 2.5 and 3.0 kg were anesthetized
by intramuscular injection of 0.5-0.7 mL/kg rodent cocktail (100
mg/mL ketamine, 20 mg/mL xylazine, and 10 mg/mL acepromazine). One
drop of proparacaine hydrochloride (0.5% Ophthaine, Bristol-Myers
Squibb) was applied to the eye prior to injection. Twenty
microliters of bacterial suspension (1.times.10.sup.2 CFU) prepared
as described above was injected into the central corneal stroma of
a randomly assigned eye while the other eye remained naive.
Injections, simulating perforation of the corneal epithelium, were
performed using a 30-gauge 1/2-inch needle and a 100 .mu.L
syringe.
[0053] For the first series of experiments, a 5-day dosing regimen
of BPI protein product (test drug) was as follows: on Day 0 of the
study, 40 .mu.L of test drug or vehicle control was delivered to
the test eye at 2 hours (-2) and 1 hour (-1) prior to intrastromal
bacterial injection (time 0), then at each of the following 10
hours (0 through +9 hrs) post-injection for a total of 12 doses (40
.mu.L/dose); on each of Days 1-4 of the study, 40 .mu.L of test
drug or vehicle control was delivered to the test eye at each of 10
hours (given at the same time each day, e.g., 8am-5 pm). For Expt.
1, 9 animals were treated, 5 with rBPI.sub.42 (1 mg/mL in 5 mM
citrate, 150 mM NaCl, 0.1% poloxamer 188, 0.002% polysorbate 80)
and 4 with buffered vehicle (5 mM citrate, 150 mM NaCl, 0.2%
poloxamer 188, 0.002% polysorbate 80). For Expt. 2, 10 animals were
treated, 5 with rBPI.sub.21 (2 mg/mL in 5 mM citrate, 150 mM NaCl,
0.2% poloxamer 188, 0.002% polysorbate 80) and 5 with buffered
vehicle. For each of Expt. 3 and Expt. 4, 5 animals were treated
with XMP.112 (1 mg/mL in 150 mM NaCl) and XMP.105 (1 mg/mL in 150
mM NaCl), respectively, and 5 animals with buffered vehicle. For
Expt. 5, 5 animals were treated with rBPI.sub.21 (2 mg/mL in 5 mM
citrate, 150 mM NaCl, 0.2% poloxamer 403, 0.002% polysorbate 80)
and 5 animals with placebo (5 mM citrate, 150 mM NaCl, 0.2%
poloxamer 403, 0.002% polysorbate 80).
[0054] For these experiments, eye examinations were conducted two
times each day for each 5-day study via slit lamp biomicroscopy to
note clinical manifestations. Conjunctival hyperemia, chemosis and
tearing, mucous discharge were graded. The grading scale for
hyperemia was: 0 (none); 1 (mild); 2 (moderate); and 3 (severe).
The scale for grading chemosis was: 0 (none); 1 (visible in slit
lamp); 2 (moderate separation); and 3 (severe ballooning). The
scale for grading mucous discharge was: 0 (none) 1 slight
accumulation); 2 (thickened discharge); and 3 (discrete strands).
Photophobia was recorded as present or absent. Tearing was recorded
as present or absent. The corneal ulcer, when present, was assessed
with respect to height (mm), width (mm), and depth (% of corneal
thickness). Neovascularization was graphed with respect to the
affected corneal meridians. Photodocumentation was performed daily
as symptoms progressed throughout the experimental procedure.
[0055] At the completion of the 5-day study period, all rabbits
were sacrificed via a lethal dose of sodium pentobarbital (6
grs/mL). Corneas were harvested and fixed in half-strength
Kamovsky's fixative. The corneas were processed for light
microscopy using Gram stain to assay for the presence of microbial
organisms and using hematoxylin and eosin to assay for cellular
infiltrate.
[0056] Examinations were conducted after injection of Pseudomonas
at 4, 24, 28, 48, 52, 72, 76, and 96 hours for these experiments.
Additional examinations were conducted at 100 and 168 hours for
Expt 3 with XMP.112 since neovascularization progressed more slowly
in this experiment than it did in others. The results of these
examinations are reported in Table 1 for Expt. 5 wherein the BPI
protein product tested (rBPI.sub.21, in a formulation with
poloxamer 403) provided the most potent effects.
1TABLE 1 Summary of Clinical Observations Neovas- Ulcer Size
Hyperemia* Chemosis* Mucous* cularization (mm) Examination
rBPI.sub.21 Plbo. rBPI.sub.21 Plbo. rBPI.sub.21 Plbo. rBPI.sub.21
Plbo. rBPI.sub.21 Plbo. Exam 1 1.2 1.0 0.2 0.3 0.5 0 None None 1
ulcer 1.4 4 hours 2 mm Exam 2 0.9 1.6 0.2 1.0 0.3 0.5 None None 1
ulcer 3.4 24 hours 6 mm Exam 3 0.6 1.7 0.2 1.1 0.6 1.3 None None 1
ulcer 5.2 28 hours 7 mm Exam 4 0.6 2.4 0.2 1.3 0.4 2.1 None None 1
ulcer 11.4 48 hours 12 mm 3 melt 1 melt 1 thinning Exam 5 0.8 2.4
0.2 1.2 0.2 1.6 None Yes 1 ulcer 11.4 52 hours (1/5) 12 mm 3 melt 1
melt 1 thinning Exam 6 0.6 2.4 0 0.8 0.2 1.0 None Yes 1 ulcer 11.4
72 hours (1/5) 12 mm 4 melt melt & thin 1 thinning Exam 7 0.6
2.4 0 0.2 0.2 0.8 None Yes 1 ulcer 11.4 76 hours (2/5) 12 mm 4 melt
melt & thin 3 thinning Exam 8 0.6 2.4 0 0.2 0.2 0.8 None Yes 1
ulcer 11.4 96 hours (2/5) 12 mm 4 melt melt & thin 3 thinning
*Mean scores of clinical observations graded on a scale of 0 (none)
to 3 (severe).
[0057] The results set out in Table 1 reveal that treatment of the
eye prior to and after perforation injury and injection of
Pseudomonas provided substantial benefits in terms of reduced
hyperemia, chemosis and mucous formation, as well as reduction in
incidence of neovascularization along with reduced incidence and
severity of corneal ulceration. At four hours after Pseudomonus
injection, fluorescein staining of the cornea in both treated and
control animals revealed small areas of staining consistent with
the injection (puncture) injury. At 28 hours after injection, the
rBPI.sub.21 treated eye evidenced clear ocular surfaces and
typically were free of evidence of hyperemia, chemosis and mucous
discharge while the vehicle treated eyes showed clouding of the
ocular surface resulting from corneal edema and infiltration of
white cells. Iritis was conspicuous in the vehicle treated eyes at
28 hours after injection and fluorescein dye application typically
revealed areas of devitalized epithelium; severe hyperemia and
moderate to severe chemosis and mucous discharge were additionally
noted. At 48 hours after injection, mild hyperemia was sometimes
noted in the rBPI.sub.21 treated eyes but mucous discharge and
chemosis were absent; the rBPI.sub.21 treated corneas were
otherwise typically clear and healthy appearing, as evidenced by
the application of fluorescein dye. Vehicle treated eyes at 48
hours post infection displayed severe hyperemia, chemosis and
mucous discharge were present; some corneas displayed corneal
melting and thinning along with an ulcerating area clouded as a
result of edema, cellular infiltration and fibrin deposition. At 52
hours following injection, rBPI.sub.21 treated eyes exhibited clear
and healthy corneas which resisted staining with fluorescein,
indicating that the formulation is safe and non-toxic to the
corneal epithelium. In vehicle treated eyes at 52 hours post
infection, sloughing of corneal epithelium was evident and while
chemosis was decreasing, hyperemia was severe. In these
experiments, several vehicle treated eyes presented with
neovascularization, with vessels growing inward toward the central
cornea. This manifestation was not noted in any rBPI.sub.21 treated
eye.
[0058] Pathohistological evaluation of the rBPI.sub.21 treated
corneas stained with hematoxylin and eosin revealed healthy, intact
corneal epithelium and stroma; the tissue was free of white cell
infiltration. In contrast, evaluation of the vehicle treated
corneas revealed absence of an epithelium and extensive
infiltration of white cells into the corneal stroma.
[0059] Additional pathohistological evaluation of the rBPI.sub.21
treated corneas stained with toluidine blue also revealed healthy,
intact corneal epithelium and stoma, and further revealed corneal
tissue free of Pseudomonas organisms. In contrast, evaluation of
the vehicle treated corneas revealed rod shaped Pseudomonas
organisms in the tissue and the presence of white cells advancing
toward the organisms in the tissue. These results indicate
effective corneal penetration of the rBPI.sub.21 and effective
sterilization of the tissue without neovascularization.
[0060] FIGS. 1 and 2 respectively provide a photographic comparison
of representative control (placebo) and treated
(rBPI.sub.21/poloxamer 403) results at 72 hours. The fluorescein
stained treated eye (FIG. 2) is healthy and clear; no keratitis is
evident, confining safety of chronic use in rabbits. In the
"control" eye shown, the perithelium has severely melted; the
thinning central cornea is ready to perforate. Severe hyperemia and
moderate mucous discharge is apparent. Chemosis was not
evident.
[0061] The rBPI.sub.21 formulation with poloxamer 403 tested in
these experiments achieved the most dramatic beneficial
antimicrobial and anti-angiogenic effects when compared with those
of the other BPI protein product formulations tested in this severe
Pseudomonas injury/infection rabbit model. Benefits in terms of
suppression of neovascularization were noted for treatment with the
rBPI.sub.42, rBPI.sub.21 (with poloxamer 188) and XMP.112
preparations whereas treatment with XMP.105 resulted in one of the
five treated eyes showing neovascularization as opposed to none for
the vehicle treated animals. Further, no significant effects in
reduction of hyperemia, chemosis, mucous formation and tearing were
noted. The contrast in efficacy of the BPI.sub.21/poloxamer 403
results (Expt. 5) with the lesser efficacy of the other products
and formulations in that study suggested that formulation
components, dosage and dosage regimen for a particular BPI protein
product may all have a significant role in optimizing beneficial
effects associated with practice of the invention.
[0062] The following Example illustrates practice of routine
procedures designed to assess, in part, effects of formulation
components and dosage regimens on optimization of beneficial
effects attending practice of the present invention.
EXAMPLE 2
Effect of BPI Protein product formulations and Dosing on
Pseudomonas Infection in a Corneal Ulceration Rabbit Model
[0063] The effect of BPI protein product administration following
Pseudomonas infection was evaluated in a corneal
infection/ulceration rabbit model using rBPI.sub.21 in various
formulations with (A) poloxamer 188, (B) poloxamer 333, and (C)
poloxamer 403 (as in Expt. 5 of Example 1).
[0064] For these experiments, the infectious organism was a strain
of Pseudomonas aeruginosa 19660 prepared and used to inject rabbits
as described in Example 1. In a first set of studies, no beneficial
effects were observed when the test product dosing regimen included
no pre-injection doses of BPI protein product and treatment was
withheld until commencement of ulcer formation at about 12-16 hours
after the bacterial injection. Briefly put, the dosing regimen of
BPI protein product employed was not sufficient to overcome the
massive destructive effects of large numbers of microorganisms,
where the infection was allowed to develop for 12-16 hours before
intervention.
[0065] In a second variant dosing and formulation study, the dosing
regimen was as described in Example 1 except that animals were not
dosed at 2 hours and 1 hour prior to injection with Pseudomonas,
but were dosed at the time of injection and then each hour for 12
hours on the first day of the 5 day experiment. Treatment was as in
Example 1 for days 2-5. For these experiments, animals were treated
as follows: 5 with rBPI.sub.21 formulated with poloxamer 188
(formulation A: 2 mg/mL rBPI.sub.21 in 5 mM citrate, 150 mM NaCl,
0.2% poloxamer 188, 0.002% polysorbate 80), 5 with rBPI.sub.21
formulated with poloxamer 333 (formulation B: 2 mg/mL rBPI.sub.21
in 5 mM citrate, 150 mM NaCl, 0.2% poloxamer 333, 0.002 polysorbate
80), 5 with rBPI.sub.21 formulated with poloxamer 403 (formulation
C: 2 mg/mL rBPI.sub.21 in 5 mM citrate, 150 mM NaCl, 0.2% poloxamer
403, 0.002% polysorbate 80) and 5 with phosphate buffered saline
(PBS) control. Eye examinations were carried out as described in
Example 1 and the animals sacrificed at the end of the 5 day
protocol.
[0066] Formulation C treated eyes exhibited less hyperemia than
saline treated eyes up to the 28 hour evaluation. The effect was
less at the 28 hour evaluation, while subsequent hyperemia scores
were equivalent between test and control groups. Formulation C also
consistently presented lower hyperemia scores than formulation A
and B, suggesting that eyes treated with formulation C were not
eliciting as much of an inflammatory response as observed the eyes
in the other treated groups.
[0067] Formulation C also elicited significantly lower scores for
chemosis than control at the 28 hour evaluation. This effect was
less at the 24 hour evaluation. Clinical scores for chemosis were
consistently lower for group C than any of the other treated
groups. As hyperemia increases, the vessels become progressively
permeable, allowing increased serum deposition into the tissues.
The formulation C treated eyes, which elicited the lowest degree of
hyperemia, presented the lowest degree of chemosis.
[0068] During the first 28 hours of the study, formulation C
treated eyes presented consistently lower mucous discharge scores
than all other groups. Neutrophil containing mucous is generally
produced in response to irritation. Control treated eyes produced
markedly greater mucous discharge during the first 28 hours of the
study than any of the active treated groups, indicating a high
degree of distress.
[0069] Formulation C treated eyes displayed the smallest ulcers
during the first 28 hours of the study, and in accordance with the
other clinical data, was the most effective antimicrobial agent of
the three formulations tested. Formulation B achieved beneficial
results superior to formulation A with respect to bactericidal
capability, although the differences were less than that between
formulations A and C. All eyes, however, were overwhelmed by the
Pseudomonas over the 28 to 48 hour period.
[0070] In these experiments, formulation C demonstrated potent
antimicrobial properties and was able to suppress ulcer
progression.
EXAMPLE 3
Effect of Administration of BPI Protein Product and Antibiotic for
Pseudomonas Infection in a Corneal Ulceration Rabbit Model
[0071] The effect of BPI protein product administration for
Pseudomonas infection is evaluated in a corneal
infection/ulceration rabbit model using a BPI protein product, such
as rBPI.sub.21, in various formulations alone and in
co-administration with various antibiotics. Experiments are
performed as described in Examples 1 and 2, but wherein the BPI
protein product is administered as an adjunct to antibiotic
treatment. Experiments are performed as described in Examples 1 and
2, except that antibiotic dosing is performed in additional to
dosing with BPI protein product. For these experiments, the
antibiotic dose is administered before, simultaneously with, or
after each dose of BPI protein product.
[0072] Numerous modifications and variations of the above-described
invention are expected to occur to those of skill in the art.
Accordingly, only such limitations as appear in the appended claims
should be placed thereon.
Sequence CWU 1
1
* * * * *